Dioxins or dioxin-like compounds are environmental pollutants produced as unwanted byproducts of common industrial processes such as paper bleaching, incineration and chemical manufacturing. Dioxin-like compounds often occur as poorly defined mixtures of these compounds in a larger matrix of other materials that make their analysis and quantitation difficult. There is considerable interest in the study, detection, monitoring and bioremediation of the compounds due to their environmental persistence, extreme chemical stability and extreme multiple toxicities to many organisms.
Dioxins or dioxin-like compounds are a loosely defined family of organochlorine molecules with close structural and chemical similarities. Additionally, these compounds, by virtue of their similar structure and chemistry, share a common mechanism of toxicity. The prototypical dioxin, and the best studied, is 2,3,7,8 tetrachlorodibenzo-p-dioxin (sometimes called 2,3,7,8-TCDD or TCDD or dioxin). Besides 2,3,7,8 tetrachlorodibenzo-p-dioxin, this group of compounds include not only the dibenzo-p-dioxins, but also dibenzofurans, azobenzenes, dibenzo-ethers, certain polychlorinated biphenyls, certain polyaromatics and other compounds. Toxicity of these compounds is dependent on a planar, polyaromatic structure with lateral halogen substitutions.
The biochemical and physiological basis of dioxin toxicity has been the subject of intense scientific scrutiny. Animals vary in their susceptibility to dioxins and in their symptoms. In guinea pigs, as little as 600 ng per kg produces a lethal wasting syndrome. In humans, toxic responses to dioxin exposure include several proliferative aberrations such as hyperkerotinosis and hyperplasia. Despite much research in the area, the biochemical and physiological events that produce toxicity are poorly understood.
Although the ultimate physiological events that produce toxicity are poorly understood, it is generally agreed that toxicity of these chemically and structurally related dioxin-like compounds is due to their ability, by virtue of their chemical and structural properties, to bind to the intracellular Aryl-Hydrocarbon (Ah) receptor. Although the ability of a compound to be a ligand of the Ah receptor is a requirement for dioxin-like toxicity, these compounds must also be able to promote transformation of the receptor to a DNA-binding form subsequent to ligand binding in order to be toxic. The transformation of the Ah receptor comprises a series of poorly understood events that include dissociation of the inactive receptor from a complex of proteins that include one or more molecules of the chaperonin HSP90, the formation of a new complex that includes HSP90-dissociated Ah receptor plus bound dioxin and the nuclear protein ARNT, and the binding of the Ah receptor/ARNT complex to specific DNA sequences.
These sequences, called Dioxin-Response Elements (DREs) or Xenobiotic-Response Elements (XREs), lie upstream of the promoter regions of certain genes, the most studied being the P4501AI gene. The binding of the transformed Ah receptor and associated protein(s) to the DREs enhance transcription of the associated genes. The inappropriate expression of these genes are thought to be the early events in the pleiotropic toxic response to dioxins. Alternatively, dissociation of the Ah receptor from the HSP90 complex, caused by the binding of a dioxin, may free a bound kinase and initiate important intracellular protein phosphorylation events that derange cell homeostasis which manifests as toxicity. In either hypothesis, it is fundamental that dioxins, in order to be toxic, must be able to both bind to the Ah receptor and transform it into an active form, and that this binding/transformation couplet is the central and only defined biochemical event in the pleiotropic toxic effects of dioxins.
Different dioxin-like compounds, although they share a common mechanism of toxicity, have different toxic potencies that can differ by several orders of magnitude. The toxicity of an unknown mixture of dioxin-like compounds can vary considerably depending on the identity and concentrations of the congeners present. Thus, the concept of Toxic Equivalency Factors (TEFs) and Toxic Equivalence (TEQs) have been advanced by some scientists. TEFs are the fractional toxicity of a dioxin-like compounds compared to the most toxic, prototypical 2,3,7,8-TCDD. Published TEFs are arbitrarily assigned values based on consensus toxicities in the scientific literature. TEQs are the estimated toxic potential of a mixture of these compounds calculated by adding their respective TEFs with adjustment for their respective concentrations. TEFs and TEQs have been promoted by the EPA in order to facilitate their risk and hazard assessment of these compounds when they occur as mixtures.
Dioxin-like compounds are commonly detected by extraction from the matrix, which can be a solid, fluid or gas, followed by several chromatographic clean-up steps to remove interfering compounds, and assayed by physico-chemical means, such as gas chromatography and mass spectrometry. The quantified dioxins are then converted to a TEQ by a mathematical formula which relies on the assigned TEFs of the detected congeners and their determined concentrations. To simultaneously measure the seventeen toxic congeners of dibenzo-p-dioxin and dibenzofuran alone by these methods is extremely challenging, is time-consuming and expensive and requires delicate instrumentation and highly trained personnel. (Clement, R. E. Ultratrace dioxin and dibenzofuran analysis: 30 years of advances. Analytical Chemistry 63:1130-1137, 1991).
The difficulty in determining all seventeen toxic dioxin and furan congeners of dioxin have led to several innovations in the detection of these compounds. Detection of dioxin by competitive immunoassay operates as any other competitive immunoassay for small molecules (Vanderlann, M., Stanker, L. H. and Watkins, B. E. Improvement and application of an immunoassay for screening environmental samples for dioxin contamination. Environ.Toxicol.Chem. 7:859-870, 1988). For immunoassays, high specificity for the target molecule is required in order to reduce quantitation errors. However, in this application a specific immunoassay for dioxin would not detect the other toxic congeners. If an immunoassay for dioxin could be purposefully constructed to nonspecifically detect all the toxic congeners of dioxin without detecting the non-toxic ones, there would still be no ability to distinguish the relative toxicity of the mixture detected.
Utilization of ability of the Ah receptor to bind dioxin has been used to construct a competitive binding assay where TCDD is presented to a mixture of mouse cytosol containing HSP90-complexed Ah receptor and a radiolabled dioxin analogue (Bradfield, C. A. and Poland, A. A competitive binding assay for 2,3,7,8-tetrachlorodibenzo-p-dioxin and related ligands of the Ah receptor. Mol.Pharmacol. 34:682-688, 1988). After a period of incubation where unknown dioxin and radiolabled dioxin analogue compete for a limited number of Ah receptor binding sites, the unbound radiolabled dioxin analogue is precipitated and the bound radioactivity counted. The number of counts is inversely proportional to the amount of TCDD present in the sample. The assay requires that the Ah receptor is maintained in a state where it can bind TCDD but not transform. The authors suggest the assay could be used to screen for other ligands of the Ah receptor but do not teach its use to estimate TEFs or TEQs. Indeed, the correlation between TEFs and receptor binding alone is poor and antagonists of the Ah receptor are known which are not toxic since they bind to the receptor but do not transform it.
Bioassays have been constructed to estimate TEQs of complex mixtures that rely on the known correlation between toxicity of a dioxin congener and it's ability to induce P4501AI enzyme activity (Tillitt, D. E., Giesy, J. P. and Ankley, G. T. Characterization of the H4IIE rat hepatoma cell bioassay as a tool for assessing toxic potency of planar halogenated hydrocarbons (PHHs) in environmental samples. Environmental Science and Technology 25:87-92, 1991). These assays require the maintenance of a cell culture which is presented with the test mixture. After an appropriate waiting period the P4501AI is extracted from the cells and its enzymatic activity measured by well known techniques. Recently, assays that use a more active and more easily measured enzyme (luciferase) have been reported (Postlind, H., Vu, T. P., Tukey, R. H. and Quattrochi, L. C. Response of human CYP1-luceferase plasmids to 2,3,7,8-tetrachlorodibenzo-p-dioxin and polycyclic aromatic hydrocarbons. Toxicol.Appl.Pharmacol. 118:255-262, 1993). In these assays an artificial DNA construct containing the promoter region of P4501AI attached to firefly luciferase are introduced into a cell line. In a similar manner, test mixture is presented to the cells and after a waiting period, the luciferase enzyme activity is measured.
The gel mobility shift assay, also called the gel retardation assay or gel shift assay, which detects the change in mobility of DNA when it interacts with protein(s), is the preferred method among scientists to detect transformation of the Ah receptor for research purposes (Denison, M. S., Fisher, J. M. and Whitlock, J. P., Jr. Protein-DNA interactions at recognition sites for the dioxin-Ah receptor complex. Journal of Biological Chemistry 264:16478-16482, 1989). In this method DREs are radiolabled, incubated with a mixture containing transformed receptor, and resolved by non-denaturing gel electrophoresis. The radiolabled DRE bands are then detected by autoradiography. The gel shift mobility assay and its sister technique, DNA footprinting, are widely used in molecular biology to study DNA-protein interactions. In gel mobility shift assays the DNA is the detected species, so they do not identify the bound protein and the identity of the bound protein can only be inferred. The single detection species in the assay prohibits the assays use when other bound proteins co-migrate with the protein of interest. Also, the assay cannot be used if the DNA-protein complex is unstable under the electrophoresis conditions. Gel chromatography has been proposed as substitution for gel electrophoresis to resolve the protein/DNA complex when the DNA-protein complex is unstable under the electrophoresis conditions, the DNA remains the detected species. Rather than curing the deficiencies of the gel shift assay, gel chromatography of the DNA/protein complex exacerbates two problems with gel shift assays. The resolution of gel electrophoresis is excellent, while gel chromatography is worse, so that any closely migrating DNA/protein complexes on gel shift assay may not be resolved at all on gel chromatography. In addition, while gel shift assays allow the side-by-side comparison of several samples, since the gel typically accommodates 10-20 lanes, only one sample per run can be injected on a gel chromatography column, eliminating side-by-side comparisons and reducing sample throughput.
Although the clinical significance of detecting DNA is well known and the clinical need to detect various proteins is well known, there is no appreciation for the clinical detection of DNA-binding proteins except autoantibodies to DNA.